With a definition for the generation of new blood vessels in adult tissues, but not for the vasculogenesis during embryogenesis or development, angiogenesis is a biological process in which angiogenic factors, substrate molecules and accessory cells are elaborately synchronized in time and space. The generation of blood vessels is achieved in complex, collective, multi-step bioreactions, playing a very important role in normal physiological functions, such as wound healing, embryogenesis, etc. In the body, angiogenesis is conducted at a necessary time in a necessary place for a required period under an elaborate system controlled by the balance between angiogenic factors and antiangiogenic factors (Loitta, L. A. et al., Cell, 64, 327 (1991)).
A failure in controlling the elaborate mechanism of angiogenesis results in various diseases, including cancers, diabetic retinopathy, rheumatoid arthritis, etc (Kohn, E. C. et al., Proc. Natl. Acad. Sci. USA, 92, 1307 (1995); Folkman, J. et al., Science, 235, 442 (1997); Risau, W., Nature, 386, 671 (1997)). Also, angiogenesis is revealed to be indispensable for the growth and metastasis of cancer cells because it enables nutrients to be provided to cancer cells and makes passageways through which cancer cells are transferred to other sites (Hanahan, D. et al., Cell, 86, 353 (1996); Skobe, M. et al., Nature Med., 3, 1222 (1997)). In detail, cancerous cells grow to the size of 2 mm or larger, new blood vessels are formed around the tumor through which the supply of oxygen and nutrients and the removal of waste products are allowed (Fidler, I. J. et al., Cell, 79, 185 (1994)). In addition, the metastasis of cancerous cells can be accelerated through the vast capillary networks newly formed by various angiogenic factors secreted from cancerous cells or normal tissue cells (Biood, C. H. et al., Biochem. Biophys. Acta., 1032, 89 (1990)).
A limitation of conventional anticancer agents and chemical therapies is that various types of cancerous cells are present even in a single tumor and they have varying mutation and growth rates that are higher than those of normal cells and consequently they become resistant to the conventional anticancer agents. In contrast, anti-angiogenic therapies for cancer inhibit the growth of host normal cells (vascular endothelial cells), but not cancerous cells themselves, so that they are expected to overcome the problems of conventional therapies for cancer, which are due to the versatility and resistance of cancerous cells. Advantages of the antiangiogenic therapies to preexisting therapies for cancer is supported by various animal test results published by many researchers (Burrows, F. J. et al., Pharmac. Ther., 64, 155 (1994)).
In the body coexist angiogenic factors and antiangiogenic factors, through the balance of which angiogenesis is elaborately performed. Until now, there have been known dozens of cancer-relevant angiogenic factors, most of which do not act as growth factors for endothelial cells (Bussolino, F. et al., Trends. Biochem. Sci. 22, 251 (1997)). On the other hand, VEGF is known to act as an endothelial cell-specific growth factor in vitro (Gospodarowics, D, et al., Proc. Natl, Acad. Sci., USA, 86, 7311 (1989)), increases vascular permeability (Leung, D. W. et al., Science, 246, 1306 (1989), and induces the angiogenesis related to the progress of cancer in vivo (Plouet, J. et al., EMBO J., 8, 3801 (1989)).
It is revealed that VEGF is one of the most potent, angiogenic factors, whose expression is induced by a variety of stimuli, including hypoxia, and is indispensably required for the growth and metastasis of human cancerous cells in vivo (Connolly, D. T. et al., J. Biol. Chem. 264, 20017 (1989); Kim, K. J. et al., Nature 362, 841 (1993)). VEGF binds to heparin and shares homology in amino acid sequence with PLGF (placental growth factor) and PDGF (platelet-derived growth factor) (Conn, G. et al., Proc. Natl. Acad. Sci., USA, 87, 2628 (1990); Keck, P. I. et al., Science, 246, 1309 (1989); Maglione, D. et al., Proc. Natl. Acad. Sci., USA, 88, 9267 (1991)). Also, it is known that VEGF is expressed as four isoforms consisting of 121, 165, 189, and 206 amino acids, respectively, by alternative splicing (Tischer, E. et al., J. Biol. Chem., 266, 11947 (1991)), of which the VEGF121 is not associated with heparin.
The signal transduction pathway of VEGF by which it exerts its functions as a growth factor starts with the binding of VEGF to cellular receptors (KDR/Flk-1 and Flt-1) which are specifically expressed on vascular endothelial cells (Millauer, B. et al., Cell, 72, 835 (1993), De Vries, C., et al., Science 255, 989 (1992)). The significance of VEGF in vasculogenesis during embryogenesis and in angiogenesis has been demonstrated by gene deletion studies of VEGF and VEGFR, (Fong, G. h. et al., Nature, 376, 66 (1995); Shalaby, F. et al., Nature, 376, 62 (1995); Carmeliet, P. et al., Nature, 380, 435 (1996); Ferrara, N. et al., Nature, 380, 439 (1996)), the malignant transformation of cancerous cells upon over-expression of VEGF in cancer cells (Zang, H. T. et al., J. Natl. Cancer Inst., 87, 213 (1995)), and the inhibition of the growth of cancer cells by neutralizing anti-VEGF monoclonal antibodies and by the expression of soluble Flt-1 and dominant-negative KDR/FlK-1 (Kim, K. J. et al., Nature, 362, 841 (1993); Goldman, C. K. et al., Proc. Natl. Acad. Sci., USA, 95, 8975 (1998)). In addition, there are reported other research results which prove that VEGF plays a key role in angiogenesis.
Therefore, materials which act to inhibit the association between VEGF and its receptors can suppress the angiogenesis driven by VEGF as well as the growth and metastasis of cancer cells, which secrete VEGF (Martiny-Baron, G. et al., Curr. Opin. Biotechnol., 6, 675 (1995)).